We propose to study the mechanisms of oxygen activation by a group of non-heme Fe-containing dioxygenases and related biosynthetic oxidases. All of the known oxygen activation strategies of are represented within this group allowing the problem to be approached on a broad front. Three of the classes of enzymes we will study are the aromatic ring cleaving, intra- and extradiol catecholic dioxygenases and the Rieske type cis-diol forming aromatic dioxygenases. These enzymes perform the key steps in the biodegradation of aromatic compounds in the environment from both natural and man-made sources; thus, they are of substantial environmental significance. Similar enzymes catalyze essential steps in mammalian biosynthetic pathways. The enzymes proposed for study in these classes include: protocatechuate 3,4- dioxygenase (3,4-PCD), homoprotocatechuate 2,3-dioxygenase (2,3-HPCD), naphthalene 1,2-dioxygenase (NDOS), and benzoate 1,2-dioxygenase (BZDOS). A second group of enzymes that will be investigated include isopenicillin N-synthase (IPNS) and aminocyclopropane-1-carboxylic acid oxidase (ACCO). IPNS catalyzes the essential formation of both rings of the penicillin class antibiotics. ACCO catalyzes synthesis of ethylene, a key hormone for plant development and ripening. These enzymes are representative of a large group of bacterial, plant, and mammalian oxidases that use oxygen to promote a specific reaction but do not incorporate O atoms into the products. We have developed hypotheses for the mechanisms by which active site iron in both the dioxygenases and the biosynthetic oxidases is used to promote catalysis through the use of several ligand positions to simultaneously bind substrates. Investigations of each enzyme class will employ a wide variety of active site mutants in conjunction with transient kinetics, spectroscopy (optical, EPR, rRaman, NMR, EXAFS, NIR CD, MCD, and Mossbauer), cryogenic techniques, and crystallography to trap and characterize intermediates in the oxygen activation and substrate oxidation processes. Past studies have lead to the development of single turnover and peroxide shunt systems for several of the enzyme classes that will be used in the investigation. Moreover, many mutations have been characterized that will allow the specific intermediates and the unique mechanistic features of the enzymes to be explored. This work should yield fundamental information about the chemistry of oxygenases, oxygen, and iron.